Not applicable.
This invention relates to methods of identifying cancerous cells and activated lymphocytes using fluorescent reagents. More specifically, it relates to the use of fluorescent reagents to identify and quantify cancerous cells and activated T and B cells. In some embodiments of the invention, the fluorescence assay is used in conjunction with a test to measure ploidy or marker expression of the cells.
In spite of numerous advances in medical research, cancer remains the second leading cause of death in the United States. In the industrialized nations, roughly one in five persons will die of cancer. Traditional modes of clinical care, such as surgical resection, radiotherapy and chemotherapy, have a significant failure rate, especially for solid tumors. Failure occurs either because the initial tumor is unresponsive, or because of recurrence due to regrowth at the original site and/or metastases. Even in cancers such as breast cancer where the mortality rate has decreased, successful intervention relies on early detection of the cancerous cells. The etiology, diagnosis and ablation of cancer remain a central focus for medical research and development.
Neoplasia resulting in benign tumors can usually be completely cured by removing the mass surgically. If a tumor becomes malignant, as manifested by invasion of surrounding tissue, it becomes much more difficult to eradicate. Once a malignant tumor metastasizes, it is much less likely to be eradicated. Early detection can help by allowing treatment in the early stages of the disease.
The three major cancers, in terms of morbidity and mortality, are colon, breast and lung. New surgical procedures offer an increased survival rate for colon cancer. Improved screening methods increase the detection of breast cancer, allowing earlier, less aggressive therapy. Numerous studies have shown that early detection increases survival and treatment options.
Excluding basal cell carcinoma, there are over one million new cases of cancer per year in the United States alone, and cancer accounts for over one half million deaths per year in this country. In the world as a whole, the five most common cancers are those of lung, stomach, breast, colon/rectum, and uterine cervix, and the total number of new cases per year is over 6 million. About half the number of people who develop cancer die of it.
Melanoma is one of the human diseases for which there is an acute need of new therapeutic modalities. It is a particularly aggressive form of skin cancer, and occurs in increased frequency in individuals with regular unguarded sun exposure. In the early disease phases, melanoma is characterized by proliferation at the dermal-epidermal junction, which soon invades adjacent tissue and metastasizes widely. Once it has metastasized, it is often impossible to extirpate and is consequently fatal. Worldwide, 70,000 patients are diagnosed with melanoma and it is responsible for 25,000 reported deaths each year. The American Cancer Society projects that by the year 2000, 1 out of every 75 Americans will be diagnosed with melanoma.
Neuroblastoma is a highly malignant tumor occurring during infancy and early childhood. Except for Wilm""s tumor, it is the most common retroperitoneal tumor in children. This tumor metastasizes early, with widespread involvement of lymph nodes, liver, bone, lung, and marrow. While the primary tumor is resolvable by resection, the recurrence rate is high.
An estimated 178,000 new cases of lung cancer were diagnosed in 1997, accounting for 13% of cancer diagnoses. An estimated 160,000 deaths due to lung cancer occurred in 1997, accounting for 29% of all cancer deaths. The one year survival rates for lung cancer have increased from 32% in 1973 to 41% in 1993, largely due to improvements in surgical techniques. The 5 year survival rate for all stages combined is only 14%. The survival rate is 48% for cases detected when the disease is still localized, but only 15% of lung cancers are discovered that early.
Small cell lung cancer is the most malignant and fastest growing form of lung cancer and accounts for 20-25% of new cases of lung cancer. Approximately 60,000 cases were diagnosed in the U.S. in 1996. The primary tumor is generally responsive to chemotherapy, but is followed by wide-spread metastasis. The median survival time at diagnosis is approximately 1 year, with a 5 year survival rate of 5-10%.
Breast cancer is one of the most common cancers and is the third leading cause of death from cancers in the United States, with an annual incidence of about 180,200 new cases among women in the United States during 1997. About 1,400 new cases of breast cancer were diagnosed in men in 1997. In industrialized nations, approximately one in eight women can expect to develop breast cancer. The overall mortality rate for breast cancer has remained unchanged since 1930. It has increased an average of 0.2% per year, but decreased in women under 65 years of age by an average of 0.3% per year. Preliminary data suggest that breast cancer mortality may be beginning to decrease, probably as a result of increased diagnoses of localized cancer and carcinoma in situ. See e.g., Marchant (1994) Contemporary Management of Breast Disease II: Breast Cancer, in: Obstetrics and Gynecology Clinics of North America 21:555-560; and Colditz (1993) Cancer Suppl. 71: 1480-1489. Approximately 44,000 deaths (44,000 women, 300 men) in 1997 occurred due to breast cancer. In women, it is the second major cause of cancer death after lung cancer. The five-year survival rate for localized breast cancer has increased from 72% in the 1940s to 97% today. If the cancer has spread regionally, however, the rate is 76%, and for women with distant metastases the rate is 20%. Survival after a diagnosis of breast cancer continues to decline beyond five years. Sixty-five percent of women diagnosed with breast cancer survive 10 years and 56% survive 15 years.
Non-Hodgkin""s B cell lymphomas are cancers of the immune system that afflicted approximately 225,000 patients in the United States in 1996. These cancers are diverse with respect to prognosis and treatment, and are generally classified into one of three grades. The median survival of the lowest grade is 6.6 years and the higher grade cancers have much lower life expectancy. Virtually all non-Hodgkin""s B cell lymphomas are incurable. New diagnoses of non-Hodgkins lymphomas have increased approximately 7% annually over the past decade, with approximately 53,000 new diagnoses in 1996. The increase is due in part to the increasing prevalence of lymphomas in the AIDS patient population.
Colon and rectal cancer accounted for an estimated 131,000 cases in 1997, including 94,000 of colon cancer and 37,000 of rectal cancer. Colorectal cancers account for about 9% of new cancer diagnoses. About 55,000 deaths due to colorectal cancer occurred in 1997, accounting for about 10% of cancer deaths. Mortality rates for colorectal cancer have fallen 32% for women and 14% for men during the past 20 years, reflecting decreasing incidence rates and increasing survival rates. However, the mortality rate in African American men continues to rise. The 1 and 5 year relative survival rates for patients with colon and rectal cancer are 82% and 61%, respectively. When colorectal cancers are detected in an early, localized stage, the 5 year survival rate is 91%; however, only 37% of colorectal cancers are discovered at that stage. After the cancer has spread regionally to involve adjacent organs or lymph nodes, the rate drops to 63%. Survival rates for persons with distant metastases is 7%. Survival continues to decline beyond 5 years, and 50% survive 10 years.
In spite of the difficulties, effective cures using anticancer drugs (alone or in combination with other treatments) have been devised for some formerly highly lethal cancers. Most notable among these are Hodgkin""s lymphoma, testicular cancer, choriocarcinoma, and some leukemias and other cancers of childhood. For several of the more common cancers, such as cervical cancer, early diagnosis, appropriate surgery or local radiotherapy enables a large proportion of patients to recover.
Cervical cancer is a relatively common, potentially lethal disease. This cancer produces few symptoms, except for irregular bleeding (usually postcoital). Cervical dysplasia most often affects women in their 20""s; carcinoma in situ affects women 30 to 39 years of age; and invasive carcinoma affects women older than 40 years old. Klemm et al. (1996) J. Obstet. Gynecol. Neonatal. Nurs. 25:629-34. Advanced lesions require surgery and possibly radiation and chemotherapy. Guzinski (1987) McGraw-Hill Encyclopedia of Science and Technology, 6th Edition, Vol. 15, p. 359. An estimated 13,500 cases of invasive cervical cancer and 6,000 related deaths occur each year in the United States, with 160,000 related deaths occurring annually worldwide. Miller et al. (1992) Am. Fam. Phys. 45:143-150; and Burger et al. (1996) J. Natl. Cancer Inst. 88:1361-8.
The standard test for cervical cancer is the Papanicolaou technique, commonly known as a Pap smear. G. N. Papanicolaou, the father of exfoliative cytology, while examining vaginal smears for cytological changes related to the menstrual cycle, discovered the presence of highly abnormal cells which proved to be malignant tumor cells. He and H. F. Traut published their first studies in 1943 on the potential of cancer diagnosis from the examination of smears of the vaginal fluid. Since the cells accumulate in the body fluids by spontaneous shedding from adjacent organs, the procedure is generally referred to as exfoliative cytology. This type of examination is applied to practically every body fluid and frequently the cellular material is enriched by mechanical abrasion such as curettage or washing. This was an extremely important step in preventative medicine; the procedure is still in use primarily as a screening test in women for the detection of early uterine cancer. Barry (1987) McGraw-Hill Encyclopedia of Science and Technology, 6th Edition, Vol. 4, p. 36. It has been said that no other test has been as successful as the Pap smear in preventing cancer. DeMay (1997) Arch. Pathol. Lab. Med. 121:229-38.
However, there are several limitations to Pap smears. Because Pap smears involve the manual histologic examination of stained cellular smears, the technology is laborious and fraught with variation in interpretation leading to the potential for false negative diagnoses. Davey (1997) Arch. Pathol. Lab. Med. 121:267-9; Mitchell et al. (1995) Cytopath 6:368-75. Other problems with the Pap smear test include severe cytotechnologist shortages, lack of internal quality controls, and problems with classification of results. Slagel et al. (1995) Diag Cytopath. 13:26-30; and de Leon-Antoni (1991) Bol. Asoc. Med. P. R. 83:462-4. Furthermore, interpretation of cytologic findings is complicated by the fact that many different classification criteria are used for a single pathological condition. In addition, inflammatory change or hormonal evaluation are not included in the system and classification is not compatible with evaluation of endometrial lesions or chorionic disease. Kashimura et al. (1993) Sangyo Ika Daigaku Zasshi 15:37-43. Pap smears may also be unreliable in detecting cervical intraepithelial neoplasia (CIN). Slawson et al. (1993) J. Fam. Pract. 36:289-93. Due to questions about the reliability of the Pap smear, a follow-up smear or colposcopy, or acetic acid wash of the cervix is usually recommended after a positive result. Slawson et al. (1992) J. Fam. Pract. 35:271-7. False negatives, of course, are not subject to subsequent screening and can lead to unnecessary disease advancement.
The problems are thought to contribute to the high false positive report, which may be as great as 11.6%. Nenning et al. (1995) Anal. Cell. Pathol. 9:61-8; and Barry (1987) McGraw-Hill Encyclopedia of Science and Technology, 6th Edition, Vol. 4, p. 36. The rate of false-negatives has been subject to considerable debate, with most studies placing the rate between 5% and 28%. Naryshkin (1997) Arch. Pathol. Lab. Med. 121:270-272; Barry (1987) McGraw-Hill Encyclopedia of Science and Technology, 6th Edition, Vol. 4, p.36; and Lieu (1996) J. Fam. Pract. 42:391-9. However, at least one study places the false negative rate as high as 56%. Noel (1989) J. Am. Board Fam. Pract. 2:156-160. It is thought that no amount of training or experience with human observers can reduce the error rate to zero. Automated Pap screening holds the promise of higher sensitivity, but no instruments to date have been approved as a sole means of primary screening. Lieu (1996).
Once cancer is detected, any number of treatments can be used. Common treatments of cervical cancer, which are often applied to other cancers, include surgery, brachytherapy, external beam radiotherapy, chemotherapy, adjuvant therapy, and combinations of these therapies. Stockle et al. (1996) J. Gynecol. Obstet. Biol. Reprod. 25:244-52; Roy et al. (1996) Gynecol. Oncol. 62:336-339; Eifel (1997) J. Surg. Oncol. 66:141-7; Ngan et al. (1989) Gynecol. Oncol. 35:259-62; Curtin et al. (1997) Surg. Oncol. Clin. N. Am. 6:813-830; and Park et al. (1997) Yonsei Med. J. 38:255-60.
An emerging area of cancer treatment is immunotherapy. There are a number of immunological strategies under development, including: 1. Adoptive immunotherapy using stimulated autologous cells of various kinds; 2. Systemic transfer of allogeneic lymphocytes; 3. Vaccination at a distant site to generate a systemic tumor-specific immune response; and 4. Implantation of immune cells directly into the tumor.
Adoptive immunotherapy is directed towards providing the patient with a level of enhanced immunity by stimulating cells ex vivo (e.g., with a tumor-associated antigen or cytokine), and then re-administering them to the patient. The cells are histocompatible with the subject, and are generally obtained from a previous autologous donation. Zarling et al. (1978) Nature 274:269-71; U.S. Pat. Nos. 5,192,537; 5,308,626; Rosenberg (1987) New Engl. J. Med. 316:889-897; Merchant et al. (1988) Cancer 62:665-671; Merchant et al. (1990) J. Neuro-Oncol. 8:173-198; and Rosenberg et al. (1990) New Engl. J. Med. 323:570-578.
In systematic (or adoptive) transfer of allogeneic lymphocytes, the goal is to create a general level of immune stimulation, thereby overcoming the anergy that prevents the host""s immune system from rejecting the tumor. Strausser et al. (1981) J. Immunol. 127:266-271; Zarling et al. (1978) Nature 274:269-271; and Kondo et al. (1984) Med. Hypotheses 15:241-77.
The third immunotherapy strategy is the generation of an active systemic tumor-specific immune response of host origin. This is achieved by administering a vaccine composition at a site distant from the tumor. Various types of vaccines have been proposed, including isolated tumor-antigen vaccines and anti-idiotype vaccines. Another approach is to use tumor cells from the patient, or derivatives of such cells. Schirrmacher et al. (1995) J. Cancer Res. Clin. Oncol. 121:487-489; and U.S. Pat. No. 5,484,596. In yet another approach, autologous or syngeneic tumor cells are genetically altered to produce a costimulatory molecule. Pardoll et al. (1992) Curr. Opin. Immunol. 4:619-23; Saito et al. (1994) Cancer Res. 54:3516-3520; Vieweg et al. (1994) Cancer Res. 54:1760-1765; Gastl et al. (1992) Cancer Res. 52:6229-6236; and WO 96/07433. Tumor cells have been genetically altered to produce TNF-I, IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IFN-I, IFN-K and GM-CSF.
The fourth immunotherapy strategy is intra-tumor implantation, which delivers effector cells directly to the tumor site. The proximity of the effector cells to the target is thought to promote the ability of the transplanted cells to react with the tumor, generating a graft versus tumor response. In studying adoptive immunotherapy of the 9L rat gliosarcoma cell line, Kruse et al. analyzed various effector cell populations, and found that tumor engraftment in the brain was prevented by allogeneic cytotoxic T lymphocytes prepared by coculturing thoracic duct lymphocytes from one inbred rat strain with spleen cells from rats syngeneic to the challenged animals. Kruse et al. (1990) Proc. Natl. Acad. Sci. USA 87:9377-9381; and Kruse et al. (1994) J. Neuro-Oncol. 19:161-168.
Redd et al. (1992) Cancer Immunol. Immunother. 34:349 describe a method of generating allogeneic tumor-specific cytotoxic T lymphocytes. CTL were generated in culture from an inbred rat strain allogeneic to the tumor cell line. The cells were found to lyse both tumor cells and Con A stimulated lymphoblasts of the same tissue type. The tumor-specific subset was deliberately selected and enriched as being specific for a determinant expressed only by the tumor.
More recently, Kruse et al. (Proc. Am. Assoc. Cancer Res. 36:474, 1995; FASEB J. 10:A1413, 1996) briefly outlined a clinical study of human brain cancer patients. The patient""s lymphocytes were expanded with OKT3 and IL-2, then co-cultured with allogeneic donor cells for 18-21 days in the presence of IL-2. Such culture conditions would result in a population highly enriched for terminally differentiated effector cells.
Considerable progress was made towards a simpler and more effective immunotherapeutic strategy by the development of cytoimplants. WO 95/20649. Potent cellular compositions are placed directly into the tumor bed, leading to beneficial effects for patients with different types of cancers. The method can be conducted as follows: The tumor patient""s leukocytes are co-cultured in a mixed lymphocyte cell reaction with healthy lymphocytes derived from an allogeneic donor. The alloactivated cells are surgically implanted at the tumor site, and produce a mixture of cytokines which induce a primary immune response. During this reaction, the host lymphoid cells identify both the graft lymphoid cells and tumor tissue as foreign.
In addition to the techniques referenced above, lymphocytes may be activated in vitro by contacting them with a variety of known lectins, mitogens, antigens (e.g. alloantigens), antibodies, other cells (as in MLC), or any combination of these stimulants. Cytotoxic T lymphocytes can be activated by, for example, immobilized anti-TcR monoclonal antibody, an immunocomplex of anti-TcR monoclonal antibody and immobilized rabbit anti-mouse antibody, a mixture of xcex2-phorbol-12 myristate-13 acetate and ionophore A23187, and immobilized concanavalin A. U.S. Pat. No. 5,180,662. T cells can also be activated by superantigens (e.g. viral superantigens). Schafer et al. (1995) Adv. Pediatr. Infect. Dis. 10:369-90. Supplementing the growth medium with mouse serum rather than the conventional calf serum apparently enhances the mitogen-induced proliferation of human T lymphocytes. U.S. Pat. No. 4,596,774.
Early activation events in T lymphocytes require the triggering of a tyrosine phosphorylation pathway involving one or more src family kinase. Weiss (1993) Cell 73:209-212. A limited number of these kinases, Lck, Fyn, and Yes, are expressed in T cells. Of these, the best-characterized is the lymphocyte-specific tyrosine kinase, P56 sup lck (Lck), whose unique N-terminal domain interacts with the cytoplasmic tails of the CD4 and CD8 glycoproteins. These glycoproteins bind to surface MHC class II and class I molecules, respectively, and participate with the T cell antigen receptor (TCR) in early events of T cell activation. Rudd et al. (1988) Proc. Natl. Acad. Sci. USA 85:5190-5194; Veillette et al. (1988) Cell 55:301-308; Shaw et al. (1989) Cell 59:627-636; Turner et al. (1990) Cell 60:755-765; and Shaw et al. (1990) Mol. Cell. Biol. 10:1853-1862.
Activated lymphocytes demonstrate altered expression of cellular markers. Known lymphocyte activation markers include CD25, CD30, CD38, CD44, CD45 (including isoforms CD45RA and CD45RO), CDw49b (VLA-2), CD56, CD69, CD71 (transferrin receptor), CD72, 4F2, HLA-DR, IL-2R (Interleukin-2 receptor), IRac, LFA-1 (lymphocyte activation marker), and serum neopterin. Norazmi et al. (1995) Immunol. Cell. Biol. 73:245-248; Chiba et al. (1995) J. Neurol. Sci. 132:170-173; Eskandari et al. (1997) Am. J. Physiol. 273:G727-34; Taher et al. (1996) J. Biol. Chem. 271:2863-7; Hughes et al. (1996) Am. J. Physiol 271:L79-84; Goodyear et al. (1996) Br. J. Dermatol. 134:85-93; O""Hanlon et al. (1996) Ann. Clin. Biochem. 33:536-9; Rabb et al. (1994) Am. J. Kidney Dis. 24:443-52; Carotti et al. (1994) Rheumatol. Int. 14:47-52; Engert et al. (1994) Leuk. Lymphoma 13:441-448; Yacyshyn et al. (1995) Int. J. Cancer 61:47-474; and Kawamura et al. (1991) Cell Immunol. 133:468-83. These markers demonstrate increased expression following lymphocyte activation. For example, LFA-1 levels increase after cells are induced with bacterial lipopolysaccharides, and serum soluble IL-2 receptor levels are higher in rheumatoid patients than in controls. Some of these markers have been studied for possible use in disease treatment. CD30, for example, is a target for immunotherapy of Hodgkin""s lymphoma. Schnell et al. (1995) Int. J. Cancer 63:238-44; and Barth et al. (1996) Ann. Oncol. 7 suppl. 4:135-141.
Methods of determining lymphocyte activation would be useful in examination of the potency of any given antigen, antibody, or lectin. Thus, the potential of the immune cell as a therapeutic can be assessed prior to therapy. These methods may also be used to determine if a certain drug is capable of blocking lymphocyte activation, as demonstrated in, for example, U.S. Pat. No. 5,439,819.
Several methods have been devised for determining activation of lymphocytes. In the formazan reduction test, activated cells are treated with a tetrazolium compound, which the cell converts to formazan. The conversion is directly related to cell proliferation. Typical tetrazolium compounds used are 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). Another method of determining the extent of activation is determining the presence of surface receptors and surface immunoglobulins, such as those listed above. U.S. Pat. No. 4,402,934. These tests for lymphocyte activation are less than perfect; the positive predictive value of these tests may be as low as 60% to 70%. The tetrazolium tests are subject to a myriad problems. The MTT assay often generates false positive and false negative results. Rollino et al. (1995) J. Immunol. Methods 185:141-3; Nishida et al. (1992) Hum. Cell 5:87-98; Campos et al. (1974) Arch. Intern. Med. 133: 432-6; and Ashburn et al. (1973) Blood 41:9215. The tetrazolium assay may underestimate the cytotoxicity of toxic agents and does not reliably measure blood mononuclear cell responses. te Boekhorst et al. (1993) Leukemia 7:1637-44; and Chen et al. (1990) Int. Arch. Allergy Appl. Immun. 93:249-55. The MTT calorimetric assay is also relatively insensitive and unable to quantify fewer than 20,000 cells. Givens et al. (1990) Invest. Opthalmol. Vis. Sci. 31:1856-62. Tetrazolium also presents technical difficulties, in that the formazan dye crystals are largely insoluble. Monner (1988) Immunol. Lett. 19:261-8; and Kasugai et al. (1990) Jpn. J. Pharmacol. 52:95-100.
All references cited herein are hereby incorporated by reference in their entirety.
The present invention encompasses methods of determining whether a test cell is cancerous and also determining if a lymphocyte is activated based on measurements of the activity of an esterase or other enzyme and comparison with the activity in cancerous or activated cells respectively.
Accordingly, one embodiment of the present invention is a method of determining if a cell in a biological sample is cancerous by providing a biological sample comprising at least one mammalian test cell; measuring the activity or level of a protein in the test cell, where the activity or level of the protein in a cancerous cell is altered from that in a non-cancerous cell; and comparing the activity or level of protein in the test cell to the level or activity of the protein in a non-cancerous cell, where the activity or level of the protein in the test cell altered from that in a non-cancerous cell indicates a probability that the test cell is cancerous.
Another embodiment of the present invention is a method of determining whether a cell in a biological sample is cancerous by providing a biological sample comprising at least one mammalian test cell; measuring the esterase activity of the cell; comparing the esterase activity of the cell to the esterase activity of a non-cancerous cell, where the esterase activity of the test cell elevated over the esterase activity of the non-cancerous cell indicates a probability that the test cell is cancerous.
In another embodiment of the invention, the determination of the level of esterase activity is performed by adding to said sample a first reagent comprising a composition which, when cleaved by said esterase, produces a first product capable of producing a detectable signal; and detecting and measuring the level of said signal, where said level is indicative of the level of esterase activity in the cell.
In another embodiment of the invention, the method further comprises a measurement of the ploidy of the test cell in a method by determining the DNA ploidy of the cell; and comparing the ploidy of the cell to the ploidy of a non-cancerous cell, where the ploidy of the test cell greater than the ploidy of a non-cancerous cell indicates a probability that the test cell is cancerous. The point at which ploidy is determined in the test is irrelevant.
In another embodiment of the invention, the determination of ploidy is performed by adding to the sample a second reagent capable of interacting with the chromosomal DNA and producing a detectable signal; and detecting and measuring the level of fluorescence, where the signal level is correlated to the ploidy of the cell.
The test cell can be an animal cell, preferably a human cell. The human cell includes, but is not limited to, those derived from cervical, brain, lung, liver, stomach, prostate, breast, epithelial, blood, bone, bladder, or colon sources. The biological sample can also be obtained from a cervical swab smear, or biopsy, blood or tissue sample.
The first reagent preferably comprises a composition which, when cleaved by said esterase, fluoresces. Suitable fluorescent compositions include, but are not limited to, fluorescein or a derivative of fluorescein. The derivative of fluorescein includes, but is not limited to, fluorescein diacetate, carboxyfluorescein diacetate, halogenated fluorescein diacetate, carboxylated halogenated fluorescein diacetate, sulfofluorescein diacetate, azidofluorescein diacetate, fluoroscein monoacetate, carboxyfluorescein monoacetate, halogenated fluorescein monoacetate, carboxylated halogenated fluorescein monoacetate, sulfofluorescein monoacetate, azidofluorescein monoacetate, fluorescein conjugated to a sugar, fluorescein phosphate, fluorescein isothiocyanate, fluorescein isothiocyanate diacetate, carboxylated fluorescein, halogenated fluorescein and carboxylated, halogenated fluorescein, sulfofluorescein, and azidofluorescein.
Preferably, the carboxyfluorescein includes, but is not limited to, fluorescein diester, 5-carboxyfluorescein diacetate, and 6-carboxyfluorescein diacetate.
More preferably, the carboxylated halogenated fluorescein diacetate includes, but is not limited to, dichlorofluorescein diacetate and 6-carboxy-2xe2x80x2,7xe2x80x2-dichlorofluorescein diacetate, and diacetyl-2,7-dichlorofluorescein.
The derivative of fluorescein includes, but is not limited to, fluorescein-di-xcex8-D-galactopyranoside, and fluorescein digalactoside.
In another embodiment of the invention, the second reagent is a DNA intercalating agent. Preferably, the DNA intercalating agent is ethidium bromide or propidium iodine.
Another embodiment of the invention is a method of determining if a cell in a biological sample is cancerous by providing a biological sample comprising at least one mammalian test cell; measuring the esterase activity in the cell by adding to the sample a compound which, when cleaved by the esterase, fluoresces; and detecting and measuring the fluorescence; comparing the esterase activity of the cell to the esterase activity of a non-cancerous cell, where esterase activity of the test cell elevated over the esterase activity of the non-cancerous cells indicates a probability that the test cell is cancerous.
In another embodiment of the invention, the measurement of the ploidy of the test cell by measuring the ploidy of the cell by adding to said sample a second reagent capable of interacting with the chromosomal DNA and fluorescing; detecting and measuring the level of fluorescence, where the level of fluorescence is correlated to the ploidy of the cell; and comparing the ploidy of the cell to the ploidy of a non-cancerous cell, where a ploidy of the test cell greater than the ploidy of a non-cancerous cell indicates a probability that the test cell is cancerous. Detection of ploidy and esterase activity can be performed in any order.
Another embodiment of the invention is a method of measuring activation of a lymphocyte by providing a test lymphocyte susceptible to activation by a stimulant; contacting the test cell with the stimulant under conditions suitable for lymphocyte activation; measuring the esterase activity of the test cell at least three days after performing step (b); and comparing the esterase activity of the test cell to the esterase activity of a cell which has not been activated, where esterase activity of the test cell altered from the esterase activity of a cell which has not been activated indicates a probability that the test cell has been activated.
Another embodiment of the invention is a method of measuring activation of a lymphocyte, wherein the determination of the level of esterase activity by adding to said sample a first reagent comprising a composition which, when cleaved by said esterase, produces a first product capable of producing a detectable signal; and detecting and measuring the level of said signal, where said level is indicative of the level of esterase activity in the cell.
In another embodiment, the cleavage of a fluorescein derivative is correlated with activation of the lymphocyte.
In another embodiment, the fluorescein derivative is fluorescein diacetate.
Another embodiment of the invention is a method of measuring activation of a lymphocyte, wherein the determination of activation of the test cell is further confirmed by measuring the reduction by the cell of a test compound, wherein said reduction is positively correlated to activation; and measuring the expression of an antigen, wherein expression of the antigen is positively correlated to activation.
In another embodiment, the test compound is a tetrazolium. Preferably the tetrazolium includes, but is not limited to, 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT).
In another embodiment, the antigen is selected from CD69, CD25, CD69, CD71, and 4F2. Preferably the antigen is CD69.
In another embodiment, expression of the antigen is measured by an immunoassay or quantification of mRNA production.
In another embodiment, activation is mediated by at least another stimulant selected from lectins, mitogens, antigens, antibodies, or other cells.